The long range goal of the proposed investigation is to elucidate the role that the external environment plays in regulating the structure and function of the plasma membrane. The environments that will be considered are the aqueous space on one side of a cell and a proteinaceous substratum on the other. Examples of such cells are the endothelium lining blood and lymphatic vessels, the epithelium of kidney tubules or the alveolar cells of the lung. It would be expected that the two unique environments that the cells are exposed to would mediate the formation and the maintenance of at least two biochemically and physiologically distinct regions of the plasma membrane. In order to more fully understand how a particular environment elicits or suppresses a unique function, the region of the plasma membrane involved must be selectively isolated and directly explored. Systems analogous to the above but more amenable to direct biochemical exploration are cells grown in monolayer cell culture; there is an upper region of the plasma membrane contacting the aqueous medium and a lower region adjacent to the culture dish. Two specific objectives will be pursued using this system. One is the development of a new procedure to selectively isolate the upper and lower regions of the plasma membrane. The unique properties of the membrane components will be determined and in addition, the procedures will be designed so that the inner and outer surface of each membrane region can be directly probed. The second objective is to use procedures to determine whether new proteins are synthesized or existing ones reorganized between the upper and lower membrane regions of cells as they attach, spread and grow in monolayers. The transformed epithelial cell line, HeLa cells will be used since they are easily and economically grown in suspension and can be induced to grow in monolayers.